Transcriptional and post-transcriptional regulation of retrotransposons IAP and MuERV-L affect pluripotency of mice ES cells

Background  

In the mouse, culture of embryonic stem (ES) cells may decrease their pluripotency and give rise to foetal abnormalities in recipient embryos. These abnormalities are frequently associated with both, chromosome abnormalities or epigenetic alteration of imprinting genes; however, little is known about the epigenetic stability of endogenous retrotransposable elements (REs). In our laboratory, we came across a R1 ES cell line, which at passage 27, lost the ability of germline transmission and started inducing the kinky tail phenotype in all chimeric animals produced with it.     

Inactivation of <Emphasis Type="Italic">Dicer1</Emphasis> in Steroidogenic factor 1-positive cells reveals tissue-specific requirement for <Emphasis Type="Italic">Dicer1</Emphasis>in adrenal,testis, and ovary

Background  

The synthesis of microRNA (miRNA) is a multi-step process that requires the action of the ribonuclease Dicer1. Dicer1 is responsible for the final processing of miRNA and has been implicated in cellular processes such as proliferation, apoptosis, and differentiation. Mouse embryos lacking Dicer1 die in early embryogenesis. In this study, we investigated whether Dicer1 is required for development of adrenal, testis, and ovary in mouse embryos.     

A computational screen for site selective A-to-I editing detects novel sites in neuron specific Hu proteins

Background  

Several bioinformatic approaches have previously been used to find novel sites of ADAR mediated A-to-I RNA editing in human. These studies have discovered thousands of genes that are hyper-edited in their non-coding intronic regions, especially in alu retrotransposable elements, but very few substrates that are site-selectively edited in coding regions. Known RNA edited substrates suggest, however, that site selective A-to-I editing is particularly important for normal brain development in mammals.     

Characterization of the TRBP domain required for Dicer interaction and function in RNA interference

Background  

Dicer, Ago2 and TRBP are the minimum components of the human RNA-induced silencing complex (RISC). While Dicer and Ago2 are RNases, TRBP is the double-stranded RNA binding protein (dsRBP) that loads small interfering RNA into the RISC. TRBP binds directly to Dicer through its C-terminal domain.     

A highly conserved gene island of three genes on chromosome 3B of hexaploid wheat: diverse gene function and genomic structure maintained in a tightly linked block

Background  

The complexity of the wheat genome has resulted from waves of retrotransposable element insertions. Gene deletions and disruptions generated by the fast replacement of repetitive elements in wheat have resulted in disruption of colinearity at a micro (sub-megabase) level among the cereals. In view of genomic changes that are possible within a given time span, conservation of genes between species tends to imply an important functional or regional constraint that does not permit a change in genomic structure. The ctg1034 contig completed in this paper was initially studied because it was assigned to the Sr2 resistance locus region, but detailed mapping studies subsequently assigned it to the long arm of 3B and revealed its unusual features.     

Evaluation of combinatorial <Emphasis Type="Italic">cis</Emphasis>-regulatory elements for stable gene expression in chicken cells

Background  

Recent successes in biotechnological application of birds are based on their unique physiological traits such as unlimited manipulability onto developing embryos and simple protein constituents of the eggs. However it is not likely that target protein is produced as kinetically expected because various factors affect target gene expression. Although there have been various attempts to minimize the silencing of transgenes, a generalized study that uses multiple cis-acting elements in chicken has not been made. The aim of the present study was to analyze whether various cis-acting elements can help to sustain transgene expression in chicken fibroblasts.     

Waves of genomic hitchhikers shed light on the evolution of gamebirds (Aves: Galliformes)

Background  

The phylogenetic tree of Galliformes (gamebirds, including megapodes, currassows, guinea fowl, New and Old World quails, chicken, pheasants, grouse, and turkeys) has been considerably remodeled over the last decades as new data and analytical methods became available. Analyzing presence/absence patterns of retroposed elements avoids the problems of homoplastic characters inherent in other methodologies. In gamebirds, chicken repeats 1 (CR1) are the most prevalent retroposed elements, but little is known about the activity of their various subtypes over time. Ascertaining the fixation patterns of CR1 elements would help unravel the phylogeny of gamebirds and other poorly resolved avian clades.     

Perturbations of MicroRNA Function in Mouse Dicer Mutants Produce Retinal Defects and Lead to Aberrant Axon Pathfinding at the Optic Chiasm

Background

During development axons encounter a variety of choice points where they have to make appropriate pathfinding decisions. The optic chiasm is a major decision point for retinal ganglion cell (RGC) axons en route to their target in order to ensure the correct wiring of the visual system. MicroRNAs (miRNAs) belong to the class of small non-coding RNA molecules and have been identified as important regulators of a variety of processes during embryonic development. However, their involvement in axon guidance decisions is less clear.

Methodology/Principal Findings

We report here that the early loss of Dicer, an essential protein for the maturation of miRNAs, in all cells of the forming retina and optic chiasm leads to severe phenotypes of RGC axon pathfinding at the midline. Using a conditional deletion approach in mice, we find in homozygous Dicer mutants a marked increase of ipsilateral projections, RGC axons extending outside the optic chiasm, the formation of a secondary optic tract and a substantial number of RGC axons projecting aberrantly into the contralateral eye. In addition, the mutant mice display a microphthalmia phenotype.

Conclusions

Our work demonstrates an important role of Dicer controlling the extension of RGC axons to the brain proper. It indicates that miRNAs are essential regulatory elements for mechanisms that ensure correct axon guidance decisions at the midline and thus have a central function in the establishment of circuitry during the development of the nervous system.     

Suppression of Induced microRNA-15b Prevents Rapid Loss of Cardiac Function in a Dicer Depleted Model of Cardiac Dysfunction

Background

Dicer endonuclease, critical for maturation of miRNAs, is depleted in certain forms of cardiomyopathy which results in differential expression of certain microRNAs. We sought to elucidate the mechanisms underlying the rapid loss of cardiac function following cardiac-specific Dicer depletion in adult mice.

Results

Conditional Dicer deletion in the adult murine myocardium demonstrated compromised heart function, mitochondrial dysfunction and oxidant stress. Elevated miR-15b was observed as an early response to Dicer depletion and was found to silence Pim-1 kinase, a protein responsible for maintaining mitochondrial integrity and function. Anti-miRNA based suppression of induced miRNA-15b rescued the function of Dicer-depleted adult heart and attenuated hypertrophy.

Conclusions

Anti-miRNA based suppression of inducible miRNA-15b can prevent rapid loss of cardiac function in a Dicer-depleted adult heart and can be a key approach worthy of therapeutic consideration.     

Dicer knockdown induces fibronectin-1 expression in HEK293T cells via induction of Egr1

Background

Dicer is a multidomain ribonuclease III enzyme involved in the biogenesis of microRNAs (miRNAs) and small interfering RNAs (siRNAs); depletion of Dicer was found to impair the migration of endothelial cells.

Methods

siRNA transfection, cell migration assay, real-time RT–PCR, chromatin immunoprecipitation, Western blotting, ELISA, caspase-3 activity assay, and annexin-V–FITC assay were utilized.

Results

Knockdown of Dicer impairs the migratory capacity of HEK293T cells and induces fibronectin-1. The upregulation of fibronectin-1 is dependent on Egr1. Fibronectin-1/Dicer double-knockdown cells showed a marked increase in apoptosis compared with fibronectin-1 single knockdown cells.

Conclusions

Decreased Dicer expression induces fibronectin-1 expression via an Egr1-dependent manner.

General significance

Our data suggest that upregulation of fibronectin-1 protects Dicer knockdown HEK293T cells against apoptosis.     

High-yield production of human Dicer by transfection of human HEK293-EBNA1 cells grown in suspension

Background

Dicer is a 219-kDa protein that plays key roles in gene regulation, particularly as the ribonuclease III enzyme responsible for cleaving precursor miRNA substrates. Its enzymatic activity is highly regulated by protein factors, and this regulation can impact on the levels of miRNAs and modulate the behavior of a cell. To better understand the underlying mechanisms of regulation, detailed enzymatic and structural characterization of Dicer are needed. However, these types of studies generally require several milligrams of recombinant protein, and efficient preparation of such quantities of pure human Dicer remains a challenge. To prepare large quantities of human Dicer, we have optimized transfection in HEK293-6E cells grown in suspension and streamlined a purification procedure.

Results

Transfection conditions were first optimized to achieve expression levels between 10 and 18?mg of recombinant Dicer per liter of culture. A three-step purification protocol was then developed that yields 4–9?mg of purified Dicer per liter of culture in a single day. From SEC-MALS/RI analysis and negative stain TEM, we confirmed that the purified protein is monomerically pure ( ≥ 98%) and folds with the characteristic L-shape geometry. Using an electrophoretic mobility shift assay, a dissociation constant (K_d) of 5?nM was measured for Dicer binding to pre-let-7a-1, in agreement with previous reports. However, when probing the cleavage activity of Dicer for pre-let-7a-1, we measured k_cat (7.2?±?0.5?min^??1) and K_M (1.2?±?0.3?μM) values that are much higher than previously reported due to experimental conditions that better respect the steady-state assumption.

Conclusions

The expression and purification protocols described here provide high yields of monomerically pure and active human Dicer. Cleavage studies of a pre-let-7 substrate with this purified Dicer reveal higher k_cat and K_M values than previously reported and support the current view that conformational changes are associated with substrate binding. Large quantities of highly pure Dicer will be valuable for future biochemical, biophysical and structural investigations of this key protein of the miRNA pathway.

     

Prognostic Significance of Deregulated Dicer Expression in Breast Cancer

Background

Dicer, an RNase III-type endonuclease, is the key enzyme involved in RNA interference and microRNA pathways. Aberrant expression of Dicer is reported in several human cancers. Our aim was to assess the prognostic role of Dicer in breast cancer.

Methods

The entire series comprised 666 invasive breast cancers (IBCs), 480 DCIS cases (397 associated with IBC and 83 pure DCIS) and 305 lymph node metastases. Cytoplasmic Dicer expression by immunohistochemistry was scored as negative (no staining) and positive (weak, moderate or strong staining).

Results

Dicer staining was assessable in 446 IBC, 128 DCIS and 101 lymph node metastases. Expression of Dicer was observed in 33% (145/446) of IBCs, 34% (44/128) of DCIS and 57% (58/101) of lymph node metastases. Dicer expression was increased in nodal metastases compared to primary tumours (p<0.001); and was associated with ER negativity (p<0.001), HER2 positivity (p<0.001), high Ki67 labeling index (p<0.001) and expression of basal-like biomarkers (p = 0.002). Dicer positivity was more frequent in the HER2 overexpressing (p<0.001) and basal-like (p = 0.002) subtypes compared to luminal A subtype. Dicer expression was associated with reduced overall survival (OS) on univariate analysis (p = 0.058) and remained an independent predictor of OS on multivariate analysis (HR 2.84, 95% CI 1.43–5.62, p = 0.003), with nodal status (HR 2.61, 95% CI 1.18–5.80, p = 0.018) and PR (HR 0.28, 95% CI 0.13–0.59, p = 0.001). Further, moderate or strong expression of Dicer was associated with improved disease-free survival in the HER2-overexpressing subtype compared to negative or weak expression (p = 0.038).

Conclusion

Deregulated Dicer expression is associated with aggressive tumour characteristics and is an independent prognostic factor for OS. Our findings suggest that Dicer is an important prognostic marker in breast cancer and that its prognostic role may be subtype specific.     

Comparison of chicken 7SK and U6 RNA polymerase III promoters for short hairpin RNA expression

Background  

RNA polymerase III (pol III) type 3 promoters such as U6 or 7SK are commonly used to express short-hairpin RNA (shRNA) effectors for RNA interference (RNAi). To extend the use of RNAi for studies of development using the chicken as a model system, we have developed a system for expressing shRNAs using the chicken 7SK (ch7SK) promoter.     

Molecular phylogenetics and comparative modeling of HEN1, a methyltransferase involved in plant microRNA biogenesis

Background  

Recently, HEN1 protein from Arabidopsis thaliana was discovered as an essential enzyme in plant microRNA (miRNA) biogenesis. HEN1 transfers a methyl group from S-adenosylmethionine to the 2'-OH or 3'-OH group of the last nucleotide of miRNA/miRNA* duplexes produced by the nuclease Dicer. Previously it was found that HEN1 possesses a Rossmann-fold methyltransferase (RFM) domain and a long N-terminal extension including a putative double-stranded RNA-binding motif (DSRM). However, little is known about the details of the structure and the mechanism of action of this enzyme, and about its phylogenetic origin.     

Up-regulated Dicer expression in patients with cutaneous melanoma

Background

MicroRNAs (miRNAs) are small non-coding RNAs (18–24 nucleotides) that have recently been shown to regulate gene expression during cancer progression. Dicer, a central enzyme in the multi-component miRNA biogenesis pathway, is involved in cutting precursor miRNAs to functionally mature forms. Emerging evidence shows that Dicer expression is deregulated in some human malignancies and it correlates with tumor progression, yet this role has not yet been investigated in skin cancers.

Methods and Findings

Using an anti-human monoclonal antibody against Dicer and immunohistochemistry, we compared the expression of Dicer protein among 404 clinically annotated controls and skin tumors consisting of melanocytic nevi (n = 71), a variety of melanomas (n = 223), carcinomas (n = 73) and sarcomas (n = 12). Results showed a cell-specific up-regulated Dicer in 81% of cutaneous, 80% of acrolentiginous and 96% of metastatic melanoma specimens compared to carcinoma or sarcoma specimens (P<0.0001). The expression of Dicer was significantly higher in melanomas compared to benign melanocytic nevi (P<0.0001). In patients with cutaneous melanomas, Dicer up-regulation was found to be significantly associated with an increased tumor mitotic index (P = 0.04), Breslow''s depth of invasion (P = 0.03), nodal metastasis (P = 0.04) and a higher American Joint Committee on Caner (AJCC) clinical stage (P = 0.009). Using western blot analysis, we confirmed the cell-specific up-regulation of Dicer protein in vitro. A pooled-analysis on mRNA profiling in cutaneous tumors showed up-regulation of Dicer at the RNA level in cutaneous melanoma, also showing deregulation of other enzymes that participate in the biogenesis and maturation of canonical miRNAs.

Conclusions

Increased Dicer expression may be a clinically useful biomarker for patients with cutaneous melanoma. Understanding deregulation of Dicer and its influence on miRNA maturation is needed to predict the susceptibility of melanoma patients to miRNA-based therapy in the future.     

Effects of osteoprotegerin from transfection of pcDNA3.1(+)/chOPG on bioactivity of chicken osteoclasts

Background  

Osteoprotegerin (OPG) has been reported to prevent bone resorption by inhibiting the formation, function, and survival of osteoclasts in a variety of animal models. However, the effects of OPG on bone metabolism in avian species have not been described. The objective of this study was to investigate the effects of chicken OPG (chOPG) expressed in chicken embryo fibroblasts (CEFs) on chicken osteoclast function in vitro.